Project Summary/Abstract Multiple sclerosis (MS) is an autoimmune disease that is characterized by demyelination and inflammation in the central nervous system (CNS). In 2010 the FDA approved fingolimod as the first orally available MS drug. It is phosphorylated in vivo, allowing it to bind four of the five known sphingosine 1-phosphate (S1P) receptors (S1PR), S1P1, 3, 4, 5. The mechanism of action (MOA) of fingolimod in MS is thought to be through lymphocyte sequestration of pathogenic cells in the secondary lymphoid organs via inactivation of S1P1. However, several lines of evidence including studies using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), have supported the existence of another MOA for fingolimod involving non-immune, CNS activities. We will test the central hypothesis that each S1PR has a unique role in EAE development and progression through regulating the immune and/or nervous system. We will apply an innovative approach, that utilizes nuclear RNA-seq and reporter mice that specifically detect activity after EAE challenge that has identified astrocytes, along with on a collection of S1P receptor knockout mice, to address three Specific Aims: Aim 1) Determine S1PRs that exacerbate EAE, followed by revealing their roles in EAE pathogenesis; focusing on S1P5 and S1P4; Aim 2)! Define VB12 homeostasis regulation by astrocyte S1P1 in EAE; and Aim 3)! Discover clinical relevancies of S1PR modulators in combination with VB12. This proposal will provide groundbreaking discoveries of: 1) roles of immunological and neuronal S1P5 in EAE pathogenesis, 2) new functions of S1P4 in T cell regulation and EAE development, 3) deciphering S1PR signaling complexity in T cells and oligodendrocytes, 4) a novel MOA of fingolimod that astrocyte S1P1 controls VB12 homeostasis in the CNS, and 5) a new therapeutic strategy, i.e., VB12 supplementation to fingolimod therapy. Moreover, our research demonstrates the biological and clinical significance of S1P signaling in MS and neuroimmunology, along with public health awareness of the importance of adequate vitamin B12 intake to prevent diseases and to maintain overall health.